patchy/holey bands after transfer, invasive bubbles?
Posted 03 July 2013 - 08:14 PM
1st year post doc here. I literally did hundreds of westerns for my PhD. New setup in new lab: Life/Invitrogen Xcell for midi gels, Bio-Rad transblot cell for wet transfer. I have done about 6 westerns successfully using precast Invitrogen midi 4-12% gels and Immobilon-FL pvdf membrane from Millipore. Two days ago I notice, after scanning my latest image, the bands have holes in them. ALSO, it looks like uneven transfer as the first 7 bands were equally loaded, but all bigger than the next 9, which were also equally loaded with one another. I have tried changing many things since then:
-New transfer buffer
-different transfer apparatus
-different pvdf membrane
-different transfer conditions (lower current)
-new methanol for wetting membrane
two things I haven't changed yet are filter paper and not using the Super Cooling Coil (both of which I used before with no incident)
literally NOTHING has made a difference. I am using smaller pre-cast gels with the Life/Invitrogen "Bolt" system now since I have a ton of them from a promotion, and just using pre-stained marker for test sample. The holes are quite visible in the ladder (see image) AFTER transfer (***important to note that the pre-stained ladder looks beautiful in the gel, and only gets these holes after transfer), so it saves me from really having to do anything downstream. Although when I have used other protein samples and did a Ponceau stain, the bands look even worse than the ladder (hard to see from my cell phone pic, but in the picture as well).
I'm basically out of ideas at this point. Of course each time I am VERY careful to roll out bubbles, etc. But it looks like somehow they are still getting between the gel and membrane, as sometimes when I can see them when I finish transfer (although not every time, so maybe I'm imagining this). One thing I don't like about the Transblot cell is the cassettes lock unevenly, and kind of make an oblong/spindle shape. I guess I'm going to just try using a setup from another lab, or even having them try everything post SDS-PAGE for me, to see if I'm the problem.
I have searched extensively online and haven't found much info. Most searches using the word "bubbles" just turn up the same obvious stuff about being extra careful to roll out bubbles, not how bubble might form/migrate in between the gel and membrane. Any advice or suggestions would be deeply appreciated. Thank you very much!
- Aarayza likes this
Posted 03 July 2013 - 08:19 PM
Trans blot lid (when I tried a new transfer system I used another trans blot system, but used my lid/cables)
Can't see how either would cause this, esp when voltage and everything looks fine, but I'm not ruling anything out at this point.
Posted 04 July 2013 - 02:12 AM
Posted 07 July 2013 - 07:15 PM
It's a long time since I've done Western blots but I seem to remember that we made our transfer buffer up early on the day we needed it, and I think we pre-chilled it. This may have helped get rid of any bubbles. You could also try vacuum degassing the transfer buffer. Have you checked the temperature of the transfer fluid during the transfer? Is it getting hotter than it should? Good luck
Posted 07 July 2013 - 07:29 PM
I think I finally fixed the problem. Although I did several things differently, I a think it has something to do with the interaction between SDS in transfer buffer and the precast gradient gels I was using.
Since my first post I tried several more things, all to no avail. The most perplexing thing was when I took a freshy run gel to my old lab and used everything from my old setup that has worked beautifully hundreds of times, and still had the same problem.
I talked to someone this morning and they asked if I used SDS in the transfer buffer. I said yes and they asked why, if I wasn't looking at high molecular weight proteins? My answer was "because I always have (0.05%)" which I realized wasn't really a good answer. So I made fresh transfer buffer w/o any SDS this time. I was also extra careful to cut everything to pefectly fit the gel (filter paper, membrane), used two pvdf membranes (read somewhere this can protect the one directly touching the gel from damage during the bubble roll-out step), and assembled the entire thing "underwater" in transfer buffer, which I usually don't do. Also, I had been using a super cooling coil with an aquarium pump in a bucket of water. Before I had stuck some frozen ice packs in there, which I thought made it pretty cold, but this time I just filled the entire thing with ice. When I came back after 2.5 hours (100mA constant voltage), the Trans Blot Cell was cold to the touch and the tubes beaded with condensation. That is pretty cool, Bio-Rad says the cold room doesn't really do anything because the transfer cells don't conduct well, but this was the first time I realized just how powerful the Super Cooling Coil is.
The molecular weight markers (I used 3 different ones this time) and sample protein (stained with Ponceau) look normal now. So why didn't this work in my old system? The only difference I can think of is before I always used homemade, 10% gels. Although I had used two different types of gels in my troubleshooting, they were both precaset and 4-12% gradient. So maybe there is something they don't like about SDS in the transfer buffer. Just a guess. I don't imagine I'll feel like testing this anytime soon.
Posted 08 July 2013 - 11:11 PM
Ran another gel, assembled stack while completely submerged (never used to do this, never had a problem with hand cast gels). Rolled out the bubbles (out of transfer buffer, atop the sponge) a little harder than usual. Now the ladder is bubble free after transfer.
So I guess these precast gels (Life Technology, Novex, 4-12%) have some predisposition to harboring bubbles that are difficult to see and roll out. Still a little baffled by the whole thing, but just wanted to put the information out there in case anyone else experiences this so maybe they don't have to go through the week of hell I did. I will try some real samples later this week and update how that goes.
Edited by jaxrich, 08 July 2013 - 11:13 PM.